Program Director/Principal Investigator (Last, First, Middle): Chiu, Wah 1R01 AI075208-01A1 ABSTRACT Herpesviruses are major pathogens in human populations, causing disease in normal and immuno-compromised individuals. Double-stranded DNA (dsDNA) tailed bacterial viruses and herpesviruses share surprising similarities in capsid assembly and organization. In both viral classes, a precursor (procapsid) shell is formed with the help of scaffolding proteins. During viral/phage morphogenesis, newly replicated dsDNA is inserted into the procapsid by a terminase protein complex through a unique portal vertex, which contains multiple gene products. In addition, both viruses deliver their nucleic acid into a membrane bound compartment while the emptied capsids remain outside this compartment. These similarities in assembly, structure, and infection suggest that herpesviruses and dsDNA phages arose from a common ancestor. The portal and associated proteins are critical in the transport of the DNA genome from the virion across the bacterial envelope for phage, and across the nuclear envelope for Herpesvirus. Until very recently the structures of such complexes - lacking icosahedral symmetry - could not be resolved by current structure analysis methods. Recent advances in asymmetric single particle reconstructions from electron cryo-microscopy images of the infectious virions of epsilon15 and P22 phages have revealed details of components of the portal vertex. However, the structural organization of the portal vertex before DNA entry and later when the DNA exits, remains to be determined. Our recent results with the herpesvirus capsid and virion suggest that its portal organization is also similar to that of the phage portal. The herpesvirus portal vertex may act like the phage portal vertex, and rearrange to release or deliver DNA into the cell nucleus. Such an apparatus presents a potential target for anti-viral therapies. We propose to use electron cryo-microscopy and electron cryo-tomography to visualize higher resolution features in the portal vertex in the herpesvirus virion and to determine the structural rearrangements of the phage portal vertex during the process of DNA ejection into host cells. The structures to be determined include not only the isolated virus particles but also the forms generated upon interaction with the host cell and release of DNA. The imaging and computational tools developed for these investigations should be generally applicable to the infection processes of many other viruses. The proposed studies, when combined with other biological and modeling information, will yield valuable structural information for developing anti-viral and gene delivery strategies targeted at these processes.